Figures & data
Figure 1. Schematic overview of cell-free production modes for membrane proteins. In the precipitate cell-free (P-CF) mode, membrane proteins aggregate due to the lack of hydrophobic environment. Membrane proteins can be co-translationally produced in a solubilized form by adding detergents (D-CF). Lipids can be provided either as liposomes (L-CF), or as nanodiscs (ND-CF).
Table I. Primers used for cloning.
Table II. Overview of produced human solute carriers.
Figure 2. Production of human solute carriers in LTE as shown by immunoblot and eGFP-fluorescence. Human transporters were either produced as N-terminal or C-terminal eGFP-fusion proteins in L. tarentolae cell-free system in 20 μl batch reactions, loaded onto a gel, blotted and detected via an anti-GFP antibody. Arrows indicate the position of the produced membrane proteins. Below the Western blot, eGFP-fluorescence of each sample is shown. lists the synthesized membrane proteins. Molecular masses of marker proteins are indicated (M). Negative control: no plasmid (-).
Figure 3. Solubilization of SLC17A3-eGFP produced in the P-CF mode by different detergents as shown by immunoblotting. Non-soluble membrane proteins were spun down and solubilized in different detergents with a final concentration of 2% for 2 h. Non-solubilized protein was removed by centrifugation and an equal amount of sample buffer was added. Same volumes of supernatant (S) and pellet (P) were loaded onto a 4–12% NuPAGE® gel, blotted and detected via an anti-GFP antibody. Used detergents are indicated above the corresponding lanes and listed in .
Table III. Used detergents for solubilization of P-CF produced solute carriers.
Figure 4. Cell-free production of SLC17A3-eGFP in the D-CF mode investigated by immunoblotting. Detergents were added to cell-free reaction mixes and the reaction mix was incubated at 26°C and 300 rpm over night. Reaction mixes were then centrifuged at 100,000g for 1 h. Pellets were resuspended in an equal volume of sample loading buffer. Same volumes of pellet (P) and supernatant (S) were loaded onto a 4–12% NuPAGE® gel. SLC17A3-eGFP was detected with an anti-GFP antibody. Molecular masses (M) of the marker proteins are indicated on the right side. Detergents and their final concentrations in the reaction are listed above the corresponding lanes and in .
Table IV. Used detergents for soluble production of solute carriers in D-CF.
Figure 5. Purification of SLC17A3-eGFP via anion exchange chromatography, produced in the D-CF mode as shown by immunoblotting and NativePAGE. SLC17A3 was produced in the D-CF mode, subsequently purified via HiTrap Q Sepharose FF column and detected via an anti-GFP antibody. M, molecular weight marker. 1–8: Elution fractions; (1) 0 mM KCl; (2) 20 mM KCl; (3) 50 mM KCl; (4) 150 mM KCl; (5) 250 mM KCl; (6) 350 mM KCl; (7) 500 mM KCl; (8) 800 mM KCl. (a) SDS-PAGE of elution fractions. (b) NativePAGE of elution fraction number 4.
Figure 6. Cell-free production of SLC17A3-eGFP in the L-CF mode as shown by immunoblotting. Liposomes were added to CECF reaction batches and incubated at 26°C and 750 rpm over night. L-CF reaction mixtures were separated via PD-10 columns and aliquots of the elution fractions were loaded onto a gel. The synthesis of protein was detected via an anti-GFP antibody. The addition of liposomes did not inhibit the synthesis of SLC17A3-eGFP. M, molecular weight marker. (1) heart lipid liposomes; (2) liver lipid liposomes; (3) brain lipid liposomes.
Figure 7. Nanodisc assembly investigated by gel filtration. Different MSP1D1:lipid ratios were tested for nanodisc assembly and analyzed via size exclusion chromatography (Superdex 200 column, GE Healthcare, Freiburg, Germany). MSPs were detected via absorbance at 280 nm. (a) MSP1D1:DMPC; (b) MSP1D1:POPC; (c) MSP1D1:liver; (d) MSP1D1:brain.
Figure 8. Purification of SLC17A3-eGFP, produced in the ND-CF mode as shown by eGFP-fluorescence and immunoblotting. SLC17A3 was produced in the ND-CF mode and subsequently purified via His-tagged MSP1D1 using Ni-NTA. Liver (a), Brain (b), DMPC (c), POPC (d) indicate the lipid sources, used for nanodisc formation. Negative control: No nanodiscs supplemented (-). FT, flow-through fraction; W, wash fraction; E, elution fraction; M, molecular weight marker. Asterisks indicate SLC17A3-eGFP and His-tagged MSP1D1, respectively. (f) Detection of SLC17A3-eGFP via anti-GFP antibody. (g) Detection of His-tagged MSP1D1 via anti-His antibody.
